Cyclic agonists and antagonists of C5A receptors and G Protein-coupled receptors

ABSTRACT

The present invention relates to novel cyclic or constrained acyclic compounds which modulate the activity of G protein-coupled receptors and are useful in the treatment of conditions mediated by G protein-coupled receptors, for example, inflammatory conditions.

This invention relates to novel cyclic compounds which have the abilityto modulate the activity of G protein-coupled receptors. The inventionprovides both agonists and antagonists. In preferred embodiments, theinvention provides cyclic peptidic and cyclic or non-cyclic non-peptidicantagonists or agonists of C5a. The compounds of the invention are bothpotent and selective, and are useful in the treatment of a variety ofinflammatory conditions.

BACKGROUND OF THE INVENTION

Activation of human complement, a system of plasma proteins involved inimmunological defence against infection and injury, contributessignificantly to the pathogenesis of numerous acute and chronicdiseases. In particular, the complement protein C5a has been extensivelyinvestigated. For general reviews, see Whaley (1987), and Sim (1993).Table 1 provides a summary of known roles of C5a in disease.

During host defence, the complement system of plasma proteins initiatesinflammatory and cellular immune responses to stimuli such as infectiousorganisms (bacteria, viruses, parasites), chemical or physical injury,radiation or neoplasia. Complement is activated through a complexcascade of interrelated proteolytic events that produce multiplebioactive peptides, some of which (eg. anaphylatoxins C3a and C5a)interact with cellular components to propagate inflammatory processes.Complement activation, either by the classical pathway, afterantigen-antibody (Ag/Ab) binding, or by the antibody-independentalternate pathway, ends with a terminal sequence in which protein C5 isproteolytically cleaved by C5 convertase to C5a and C5b. The latterfacilitates assembly of a “membrane attack complex” that punches holesin membranes of target cells such as bacteria, leading to leakage, lysisand cell death. Steps in the cascade are tightly regulated to avoidstepwise amplification of proteolysis by sequentially formed proteases.If these regulatory mechanisms become inefficient, protracted activationof complement can result, causing enhanced inflammatory responses as inautoimmune diseases.

Although the broad features of the complement system and its activationare known, mechanistic details remain poorly understood. A principal andvery potent mediator of inflammatory responses is the plasmaglycoprotein C5a, which interacts with specific surface receptors (C5aR)on mast cells, neutrophils, monocytes, macrophages, non-myeloid cells,and vascular endothelial cells (Gerard and Gerard, 1994). C5aR is a Gprotein-coupled receptor with seven transmembrane helices (Gerard andGerard, 1991). This receptor is one of the rhodopsin superfamily ofGTP-linked binding proteins, but differs from rhodopsin receptors inthat the receptor and G protein are linked prior to activation.

G protein-coupled receptors are prevalent throughout the human body,comprising approximately 80% of known cellular receptor types, andmediate signal transduction across the cell membrane for a very widerange of endogenous ligands. They participate in a diverse array ofphysiological and pathophysiological processes, including, but notlimited to those associated with cardiovascular, central and peripheralnervous system, reproductive, metabolic, digestive, immunoinflammatory,and growth disorders, as well as other cell-regulatory and proliferativedisorders. Agents, both agonists and antagonists, which selectivelymodulate functions of G protein-coupled receptors have importanttherapeutic applications.

C5a is one of the most potent chemotactic agents known, and resultsneutrophils and macrophages to sites of injury, alters their morphology;induces degranulation; increases calcium mobilisation, vascularpermeability (oedema) and neutrophil adhesiveness; contracts smoothmuscle; stimulates release of inflammatory mediators (includinghistamine, TNF-α, IL-1, IL-6, IL-8, prostaglandins, leukotrienes) andlysosomal enzymes; promotes formation of oxygen radicals; and enhancesantibody production (Gerard and Gerard, 1994). Overexpression orunderregulation of C5a is implicated in the pathogenesis ofimmunoinflammatory conditions such as rheumatoid arthritis, adultrespiratory distress syndrome (ARDS), systemic lupus erythematosus,tissue graft rejection, ischaemic heart disease, reperfusion injury,septic shock, psoriasis, gingivitis, atherosclerosis, Alzheimer'sdisease, lung injury and extracorporeal post-dialysis syndrome, and in avariety of other conditions, as summarised in Table 1.

TABLE 1 The Role of C5a in Disease C5a C5aR Condition/disease levelsexpression Details allergy ++ allergen challenge leads to nasal symptomsand increased C5a levels Alzheimer's ++ ++ up-regulation of the receptorin disease reactive astrocytes, microglia and endothelial cells in theCNS, complement system activated by β-amyloid ARDS/respiratory ++distress Behcet's disease ++ levels highest just prior to ocular attacksbronchial asthma ++ capillary leak ++ syndrome chronic lung ++ IncreasedC5a levels in pulmonary disease effluent fluid from mechanicallyventilated infants with chronic lung disease Churg-Strausshypersensitivity of granulocytes to C5a cystic fibrosis generation ofC5a/effects on PMNs decompression ++ increased C5a levels during stresssaturation diving diabetes type I ++ C5a generated during onset;circulating monocytes in newly diagnosed type I diabetes patients areactivated Familial Lack of C5a inactivator Mediterranean feverGuillain-Barre ++ CSF levels elevated ischaemic disease migration ofmonocytes into states/myocardial myocardium after reperfusion. infarctDamage prevented with sCR1 Kimura's disease humoral factor up-regulatesthe response of PMNs to C5a Multiple ++ increased expression of theSclerosis receptor on foamy macrophages in acute and chronic MS andfibrous astrocytes in chronic MS Meningitis C5a induces experimentalmeningitis; PMN accumulation seen in the CSF pancreatitis ++post-dialysis ++ − C5a generated via complement syndrome activation bytubing material, C5aR levels decreased on PMNs & monocytes in chronicstate preeclampsin/ ++ C5a levels in elevated at delivery HELLPpsoriasis ++ C5a levels high in scales reperfusion injury ++ inhibitedby C5 antibody retinitis ++ C5a detected in vitreous humor Rheumatoid ++elevated concentration of C5a arthritis found in synovial fluid (5-fold)and plasma (3-fold) Severe congenital − neutropenia transplant/graft ++monoclonal antibodies block the rejection damage seen with xenogenictransplant; increased levels of C5a seen in the plasma and urine ofpatients with renal graft rejection

New agents which limit the pro-inflammatory actions of C5a havepotential for inhibiting chronic inflammation, and its accompanying painand tissue damage. For these reasons, molecules which prevent C5abinding to its receptors are useful for treating chronic inflammatorydisorders driven by complement activation. Importantly, such compoundsprovide valuable new insights to mechanisms of complement-mediatedimmunity.

In another context, agonists of C5a receptors or other G protein-coupledreceptors may also be found to have therapeutic properties in conditionseither where the G protein-coupled receptor can be used as a recognitionsite for drug delivery, or where triggering of such receptors can beused to stimulate some aspect of the human immune system, for example inthe treatment of cancers, viral or parasitic infections.

One approach to the development of agonists or antagonists of C5a isthrough receptor-based design, using knowledge of the three-dimensionalstructures of C5a, its receptor C5aR, and the interactions between them.The structure of the receptor is unknown. The solution structure ofhuman C5a, a 74 amino acid peptide that is highly cationic andN-glycosylated with a 3 KDa carbohydrate at Asn64, has been determinedand is essentially a 4-helix bundle. The C-terminal end (residues 65-74,C5a₆₅₋₇₄) was found to be unstructured (Zuiderweg et al, 1989) and thisconformational flexibility in the C-terminus has made structure-functionstudies extremely difficult to interpret.

C5a has a highly ordered N-terminal core domain (residues 1-64;C5a₁₋₆₄), consisting of a compact antiparallel 4-helix bundle (residues4-12, 18-26, 32-39, 46-63) connected by loops (13-17, 27-31, 40-45), andfurther stabilised by 3 disulphide bonds (C21-Cys47, Cys22-Cys54,Cys34-Cys55).

Although the structure of the C5a receptor, C5aR, is unknown, theC5a-binding subunit of human monocyte-derived C5aR has been cloned andidentified as a G protein-coupled receptor with transmembrane helices(Gerard and Gerard, 1991). Interactions between C5a and C5aR have beenthe subject of many investigations which, in summary, suggest that C5abinds via a two-site mechanism in which the N-terminal core domain ofC5a is involved in receptor-recognition and binding, while theC-terminus is responsible for receptor activation. This mechanism isillustrated schematically in FIG. 1. The C-terminal “effector” regionalone possesses all the information necessary for signal transduction,and is thought to bind in the receptor's interhelical region (Sicilianoet al, 1994; deMartino et al, 1995).

An N-terminal interhelical positively-charged region of C5a isresponsible for receptor recognition and binding, and binds to anegatively-charged extracellular domain of C5aR (site 1), while theC-terminal “effector” region of C5a is thought to bind with theinterhelical region of the receptor (site 2), and is responsible forreceptor activation leading to signal transduction (Siciliano et al,1994).

Numerous short peptide derivatives of the C-terminus of C5a have beenfound to be agonists of C5a (Kawai et al, 1991; Kawai et al, 1992; Kohlet al, 1993; Drapeau et al, 1993; Ember et al, 1992; Sanderson et al,1994; Sanderson et al, 1995; Finch et al, 1997; Tempero et al, 1997;Konteatis et al, 1994; DeMartino et al, 1995). The structures of some ofthese agonists are shown in Table 2 below (compounds 1-6). Highmolecular weight polypeptide inhibitors of the action of C5a at itsreceptor, such as monoclonal antibodies to the C5a receptor, are alsoknown (Morgan et al, 1992).

A small molecule,N-methylphenylalanine-lysine-proline-D-cyclohexylalanine-tryptophan-D-arginine(7, MeF-K—P-dCha-W—R), is a full antagonist of the C5a receptor, with noagonist activity when tested on isolated cellular membranes (Konteatiset al, 1994) or intact whole cells. This hexapeptide was developed bymodifications of the agonist NMe-F—K—P-dCha-L-r, in which the moleculewas progressively substituted at leucine residues with substituents ofincreasing size (Cha, F, Nph and W). This had the effect of reducingagonist activity. Receptor-binding assays, performed on isolated humanneutrophil membranes, showed that the antagonist had only 0.04% relativeaffinity of C5a for the receptor (Konteatis et al, 1994). A key featureof these reports is the definition of the binding of 7 to the C5areceptor. These authors state that the C-terminal arginine is essentialfor receptor binding and antagonist activity. This is also the case inall the reports of agonist activity by small peptide analogues of theC-terminus of C5a. However, for the antagonist 7, the authors go furtherand state that

-   -   “the C-terminal carboxylate is an essential requirement for        antagonist activity and receptor binding.”

They proposed that the requirement of the carboxylate is probably theresult of its specific interaction with an arginine (Arg 206) in thereceptor (De Martino et al, 1995). This idea was supported by a greatreduction in receptor-affinity for an analogue of 7 in which theD-arginine (NH₂—CH(CO₂H)—(CH₂)₃NHC(:NH)NH₂) was replaced by agmatine(NH₂—CH₂—(CH₂)₃NHC(:NH)NH₂). In summary, De Martino et al claim that theD-arginine interacts via its guanidinium side chain with anegatively-charged amino acid side chain in the receptor. A secondinteraction between the negatively-charged C-terminal carboxylate of 7and a positively-charged side chain residue in the receptor is alsothought to occur.

We have now determined the solution structure of this hexapeptide 7 andseveral analogues, and have surprisingly found that in fact a terminalcarboxylate group is not required for binding to C5aR or for antagonistactivity, and that instead an unusual hitherto unrecognised structuralfeature, a turn conformation, is responsible for C5a antagonist oragonist binding and activity. The hexapeptide and several newstructurally related antagonists have been examined for both theirreceptor-binding affinities and antagonist activity, using intactpolymorphonuclear (PMN) cells. Our results show the hitherto unknownspecific structural requirement for the binding of C5a antagonists oragonists to the C5a receptor, which we believe to be common to ligandsfor the G protein-coupled receptor family. Our establishment of thisspecific structural requirement has enabled us to design and developimproved molecular probes of the complement system and of C5a-baseddrugs, and to design small molecules that target other G protein-coupledreceptors, which are becoming increasingly recognised as important drugtargets due to their crucial roles in signal transduction (Gprotein-coupled Receptors, IBC Biomedical Library Series, 1996).

Thus our results have enabled us to design constrained structuraltemplates which enable hydrophobic groups to be assembled into ahydrophobic array for interaction with a G protein-coupled receptor, forexample at Site 2 of the C5a receptor illustrated in FIG. 1. Suchtemplates or scaffolds, which may be cyclic or acyclic, have notheretofore been suggested for modulators of the activity of C5areceptors or other G protein-coupled receptors.

SUMMARY OF THE INVENTION

The invention provides cyclic and non-cyclic modulators of the activityof G-protein-coupled receptors.

According to a first aspect, the invention provides a compound which isan antagonist, of a G protein-coupled receptor, which has no agonistactivity, and which has a cyclic or constrained acyclic structureadapted to provide a framework of approximate dimensions as follows:

-   -   where the numerals refer to distances between C_(n) carbons of        amino acids or their analogues or derivatives, and A, B, C and D        are not necessarily on adjacent amino acids, or analogues or        derivatives thereof; and    -   where the critical amino acid side chains are designated by A,        B, C and D, or are as defined below;    -   A is any common or uncommon, basic, charged amino acid side        chain which serves to position a positively charged group in        this position, including, but not limited to the following side        chains and other mimetics of arginine side chains:        where    -   X is NCH, NNO₂, CHNO₂ or NSO₂NH₂;    -   n is an integer from 1 to 4, and    -   R is H or an alkyl, aryl, CN, NH₂ or OH group    -   B is any common or uncommon aromatic amino acid side chain which        serves to position an aromatic side-chain group in this        position, including but not limited to the indole, indole        methyl, benzyl, phenyl, naphthyl, naphthyl methyl, cinnamyl        group, or any other derivatives of these aromatic groups;    -   C is any common or uncommon hydrophobic amino acid side chain        which serves to position any alkyl, aromatic or other group in        this position, including, but not limited to D- or L-cyclohexyl        alanine (Cha), leucine, valine, isoleucine, phenylalanine,        tryptophan, or methionin    -   D is any common or uncommon aromatic amino acid which serves to        position an aromatic side-chain in this position, and has the        structure:        where Z includes but is not limited to indole, indole methyl,        benzyl, benzene, naphthyl, naphthyl methyl, or any other        derivatives of these aromatic groups, and    -   R¹ is H or any alkyl, aromatic, acyl or aromatic-acyl group        including, but not limited to methyl, ethyl, propyl, butyl,        —CO—CH₂CH₃, —CO—CH₃, —CO—CH₂CH₂CH₃, —CO—CH₂Ph, or —CO-Ph.

Preferably the G protein-coupled receptor is a C5a receptor.

Other cyclic or constrained acyclic molecules, which may be peptidic ornon-peptide in nature, can similarly be envisaged to support groups suchas A, B, C and D for interaction with a C5a receptor or other Gprotein-coupled receptor.

In one preferred embodiment, the compound has antagonist activityagainst C5aR, has no C5a agonist activity, and has the general formula:

where A is H, alkyl, aryl, NH₂, NHalkyl, N(alkyl)₂, NHaryl or NHacyl;OH, Oalkyl, Oaryl.

-   -   B is an alkyl, aryl, phenyl, benzyl, naphthyl or indole group,        or the side chain of a D- or L-amino acid selected from        phenylalanine, homophenylalanine, tryptophan, homotryptophan,        tyrosine, and homotyrosine;    -   C is the side chain of a D-, L- or homo-amino acid selected from        the group consisting of proline, alanine, leucine, valine,        isoleucine, arginine, histidine, aspartate, glutamate,        glutamine, asparagine, lysine, tyrosine, phenylalanine,        cyclohexylalanine, norleucine, tryptophan, cysteine and        methionine;    -   D is the side chain of a D- or L-amino acid selected from the        group consisting of cyclohexylalanine, homocyclohexylalanine,        leucine, norleucine, homoleucine, homonorleucine and tryptophan;

E is the side chain of a D- or L-amino acid selected from the groupconsisting of tryptophan and homotryptophan;

-   -   F is the side chain of a D- or L-amino acid selected from the        group consisting of arginine, homoarginine, lysine and        homolysine; and

X¹ is —(CH₂)_(n)NH— or (CH₂)_(n)—S—, where n is an integer of from 1 to4, preferably 2 or 3, —(CH₂)₂O—, —(CH₂)₃O—, —(CH₂)₃—, —(CH₂)₄—, or—CH₂COCHRNH—, where R is the side chain of any common or uncommon aminoacid.

For the purposes of this specification, the term “alkyl” is to be takento mean a straight, branched, or cyclic, substituted or unsubstitutedalkyl chain of 1 to 6, preferably 1 to 4 carbons. Most preferably thealkyl group is a methyl group. The term “acyl” is to be taken to mean asubstituted or unsubstituted acyl of 1 to 6, preferably 1 to 4 carbonatoms. Most preferably the acyl group is acetyl. The term “aryl” is tobe understood to mean a substituted or unsubstituted homocyclic orheterocyclic aryl group, in which the ring preferably has 5 or 6members.

A “common” amino acid is a L-amino acid selected from the groupconsisting of glycine, leucine, isoleucine, valine, alanine,phenylalanine, tyrosine, tryptophan, aspartate, asparagine, glutamate,glutamine, cysteine, methionine, arginine, lysine, proline, serine,threonine and histidine.

An “uncommon” amino acid includes, but is not restricted to, D-aminoacids, homo-amino acids, N-alkyl amino acids, dehydroamino acids,aromatic amino acids (other than phenylalanine, tyrosine andtryptophan), ortho-, meta- or para-aminobenzoic acid, ornithine,citrulline, norleucine, γ-glutamic acid, aminobutyric acid andα,α-disubstituted amino acids.

For the purposes of this specification it will be clearly understoodthat the word “comprising” means “including but not limited to”, andthat the word “comprises” has a corresponding meaning.

According to a second aspect, of the invention provides a compound whichis an agonist of G protein-coupled receptors, and which has structureIII

-   -   where the numerals refer to distances between C_(n) carbons of        amino acids or their analogues or derivatives, and A, B, C and D        are not necessarily on adjacent amino acids, or analogues or        derivatives thereof; and    -   where B is a non-aromatic amino acid, and is preferably the D-        or L-form of alanine, leucine, valine, norleucine, glutamic        acid, aspartic acid, methionine, cysteine, isoleucine, serine or        threonine,    -   and A, C and D are as defined above.

Preferably the compound is of structure IV,

-   -   where E is any amino acid other than tryptophan and        homotryptophan, for example D- or L-forms of alanine, leucine,        valine, norleucine, phenylalanine, glutamic acid, aspartic acid,        methionine, cysteine, isoleucine, serine, threonine, and F and        X¹ are as defined in Structure II. Preferably the compound is an        agonist of C5a.

According to a third aspect, the invention provides a composition,comprising a compound according to the invention together with apharmaceutically-acceptable carrier or excipient.

The compositions of the invention may be formulated for oral orparenteral use, but oral formulations are preferred. It is expected thatmost if not all compounds of the invention will be stable in thepresence of digestive enzymes. Such stability can readily be tested byroutine methods known to those skilled in the art.

Suitable formulations for administration by any desired route may beprepared by standard methods, for example by reference to well-knowntextbooks such as Remington; The Science and Practice of Pharmacy, Vol.II, 1995 (19^(th) edition), A. R. Gennaro (ed), Mack Publishing Company,Easton, Pa., or Australian Prescription Products Guide, Vol. 1, 1995(24^(th) edition) J. Thomas (ed), Australian Pharmaceutical PublishingCompany Ltd, Victoria, Australia.

In a fourth aspect, the invention provides a method of treatment of apathological condition mediated by a G protein-coupled receptor,comprising the step of administering an effective amount of a compoundof the invention to a mammal in need of such treatment.

Preferably the condition mediated by a G protein-coupled receptor is acondition mediated by a C5a receptor, and more preferably involvesoverexpression or underregulation of C5a. Such conditions include butare not limited to rheumatoid arthritis, adult respiratory distresssyndrome (ARDS), systemic lupus erythematosus, tissue graft rejection,ischaemic heart disease, reperfusion injury, septic shock, psoriasis,gingivitis, atherosclerosis, Alzheimer's disease, lung injury andextracorporeal post-dialysis syndrome.

While the invention is not in any way restricted to the treatment of anyparticular animal or species, it is particularly contemplated that thecompounds of the invention will be useful in medical treatment ofhumans, and will also be useful in veterinary treatment, particularly ofcompanion animals such as cats and dogs, livestock such as cattle,horses and sheep, and zoo animals, including large bovids, felids,ungulates and canids.

The compounds may be administered at any suitable dose and by anysuitable route. Oral administration is preferred because of its greaterconvenience and acceptability. The effective dose will depend on thenature of the condition to be treated, and the age, weight, andunderlying state of health of the individual treatment. This will be atthe discretion of the attending physician or veterinarian. Suitabledosage levels may readily be determined by trail and errorexperimentation, using methods which are well-known in the art.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a diagrammatic representation of the two-site model forbinding of C5a to its G protein-coupled receptor, C5aR. The black rodsrepresent a-helical regions, and the open cylinders represent thetransmembrane helices. Sites 1 and 2 are indicated on the figure.

FIG. 2 shows stacked plots of ¹H-NMR spectra, showing time-dependentdecay of amide NH resonances for Trp (8.10 ppm) and D-Cha (7.90 ppm)residues of 7 in d₆-DMSO containing D₂O after 10 minutes (bottom plot)and then 25, 40, 55, 70, 130, 190, 250, 385 and 520 minutes.

FIG. 3 shows backbone C, N, O atoms of twenty lowest energy minimizedNMR structures of 7 in d₆-DMSO at 24° C.)

FIG. 4 shows a schematic representation of H-bonding in the structure of7 from proton NMR spectra in d₆-DMSO.

FIG. 5a shows (a) receptor binding, as indicated by inhibition ofbinding of ¹²⁵I-C5a to human PMNs by 7 (●); 8 (Δ); 9 (▴); 12 (◯)

FIG. 5b shows C5a antagonist potency as inhibition of myeloperoxidase(MPO) release from human PMNs by: 7 (▪, n=9) and 12 (▴, n=4). FIG. 5cshows C5aR binding and antagonist potencies of 7, 15 and 17.

A-C show the effect of increasing concentrations (top to bottom) of C5aantagonists inhibiting myeloperoxidase release in human PMNs (n=3 inA-C).

A: 7 at 0, 0.1, 0.3, 1.0 μM (top to bottom)

B: 15 at 0, 0.1, 0.03, 0.1 μM (top to bottom)

C: 17 at 0, 0.01, 0.03, 0.1 μM (top to bottom)

D: Comparative affinities for PMN C5qR receptor.

Inhibition of binding of ¹²⁵I-C5a to human PMNs by 7 (top), (middle), 17(bottom). All data are means±SEM.

FIG. 6 shows receptor binding of cyclic C5a antagonists, as-shown byinhibition of binding of ¹²⁵I-C5a to human PMNs (n=5).

FIG. 7 shows superimposed structures of 7 (light, NMR structure) and 12(dark, computer modelled structure). Phe and Trp side chains are omittedfrom 12 for clarity.

FIG. 8 shows inhibition of C5a-induced neutropenia in Wistar rats by thecyclic antagonist F-[OPdChaWR] given i.v. at 1 mg/kg. Results shown fromn=3 in each group, *P<0.05 compared to C5a-treated group only. Resultsare expressed as means±SEM.

FIG. 9 shows inhibition of LPS-induced neutropenia and changes inhaematocrit induced by the cyclic antagonist F-[OPdChaWR] (0.03-10mg/kg, i.v., 10 min prior to lipopolysaccharide [LPS]) in Wistar rats.

Abscissa: time after LPS (1 mg/kg i.v. injection).

Ordinate: percent change in haematocrit (A) value or level ofcirculating polymorphonuclear (PMN) leukocytes (B) compared to timezero.

FIG. 10 shows inhibition of carrageenan-induced (Wistar) rat paw oedemaby cyclic antagonist (3D35) AcF-[OPdChaWr] (1 mg/kg single dose i.p.given 30 min prior to carrageenan). Results shown from 4 rats/group,means±SEM. Ordinate: percent change in paw volume. Abscissa: time (mins)after carrageenan injection

DETAILED DESCRIPTION OF THE INVENTION

The invention will now be described by way of reference only to thefollowing general methods and experimental examples, and to the figures.Abbreviations used herein are as follows:

BOP benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphoniumhexafluorophosphate D-Cha D-cyclohexylamine DIPEA diisopropylethylamineDMF dimethylformamide DMSO dimethylsulphoxide HBTU O-benzotriazoleN′,N′,N′,N′-tetramethyluronium hexafluoro- phosphate LPSlipopolysaccharide PMN polymorphonuclear granulocyte RMSD root meansquare deviation RP-HPLC reverse phase-high performance liquidchromatography TFA trifluoroacetic acid;

Throughout the specification conventional single-letter and three-lettercodes are used to represent amino acids.

General Methods

Protected amino acids and resins were obtained from Novabiochem. TFA,DIPEA and DMF (peptide synthesis grade) were purchased from Auspep. Allother materials were reagent grade unless otherwise stated. Preparativescale reverse-phase HPLC separations were performed on a Vydac C18reverse-phase column (2.2×25 cm), and analytical reverse-phase HPLCseparations were performed on a waters Delta-Pak PrepPak C18reverse-phase column (0.8×10 cm), using gradient mixtures of solventA=water/0.1% TFA and solvent B=water 10%/acetonitrile 90%, 0.09% TFA.The molecular weight of the peptides was determined by electrospray massspectrometry recorded on a triple quadrupole mass spectrometer (PE SCIEXAPI III), as described elsewhere (Haviland et al, 1995). ¹H-NMR spectrawere recorded on either a Bruker ARX 500 MHz or a Varian Unity 400spectrometer. Proton assignments were determined by 2D NMR experiments(DFCOSY, TOCSY, NOESY).

Non-peptidic compounds were synthesized using conventional organicchemical methods. Compounds were analysed by ¹H-NMR spectroscopy and bymass spectrometry.

Peptide Synthesis

Some representative peptide syntheses are now given. Linear peptidesequences were assembled by manual stepwise solid-phase peptidesynthesis with HBTU activation and DIEA in situ neutralisation. Bocchemistry was employed for temporary N^(α)-protection of amino acidswith two 1 min treatments with TFA for Boc group removal. The peptideswere fully deprotected and cleaved by treatment with liquid HF (10 ml;p-cresol (1 ml); −5° C.; 1-2 hr). Analytical HPLC (gradient; 0% B to 50%B over 40 min); 7, Rt=32.0 min, [M+H]⁺(calc.)=900.5,[M+H]⁺(exper.)=900.7; 8, Rt=32.2 min, [M+H]⁺(calc.)=899.6,[M+H]⁺(exper.)=899.7; 9, Rt=30.0 min, [M+H)⁺(calc.)=900.5,[M+H]⁺(exper.)=900.7; 10, Rt=23.8 min, [M+H]⁺(calc.)=860.5,[M+H]⁺(exper.) 860.5.

Structures for the peptides are shown in Table 4 below.

a) Synthesis of Cycle 11

This is a general method used for the synthesis of a wide range ofcyclic antagonists covered by this patent. For example, in the case ofcycle 11, its linear precursor peptide was synthesised by Fmoc chemistryusing HBTU/DIEA activation on an Fmoc-D-Arg(Mtr)-Want resin. Fmoc groupremoval was effected using two 1 min treatments with 50% piperidine/DMF.Cleavage and deprotection using 95% TFA/2.5% TIPS/2.5% H₂O gave theMtr-protected peptide, which was purified by RP-HPLC. Cyclization of theprotected, purified peptide using 3eq BOP and 10eq DIEA at a 1 mMconcentration in DMF stirring for 15 hr gave the cyclised product, whichwas fully deprotected using 1M TMSBr in TFA. A final RP-HPLCpurification gave the desired peptide in yields of 50% for thecyclisation. Rt=37.7 min, [M+H]⁺(calc.) 910.5, [M+H]⁺(exper.)=910.7.

b) Synthesis of Cycle 12

Cyclization of the cleaved and fully deprotected peptide was achieved bystirring a 1 mM solution in DMF with 3eq BOP and 10 eq pyridine as basefor 15 hr. A final RP-HPLC purification gave the desired peptide inyields of 22% for the cyclization. Rt=37.3 min, (M+H]+(calc.)=896.5,[M+H]+(exper.)=896.5.

NMR Structure Determination

¹H-NMR spectra were recorded for compound 7 (3 mg in 750 μl d₆-DMSO,δ2.50) referenced to solvent on a Varian Unity 400 spectrometer at 24°C. Two dimensional ¹H-NMR NOESY (relaxation delay 2.0 s, mix time 50-300ms), DFQ-COSY and TOCSY (mixing time 75 ms) experiments were acquiredand recorded in phase sensitive mode. Acquisition times=0.186 s,spectral width=5500 Hz, number of complex points (t₁ dimension)=1024 forall experiments. Data was zero-filled and Fourier transformed to 1024real points in both dimensions.

NMR data was processed using TRIAD software (Tripos Assoc.) on a SiliconGraphics Indy work station. 2D NOE cross peaks were integrated andcharacterised into strong (1.8-2.5 Å), medium (2.3-3.5 Å) and weak(3.3-5.0 Å). Preliminary three-dimensional structures were calculatedfrom upper and lower distance limit files using Diana 2.8 (69 distanceconstraints, including 27 for adjacent residues and 6 further away) withthe redundant dihedral angle constraints (REDAC) strategy. Upper andlower distance constraints were accurately calculated using MARDIGRAS.At this stage the peptide was examined for possible hydrogen bonds, andthese were added as distance constraints. The 50 lowest energy Dianastructures were subjected to restrained molecular dynamics (RMD) andenergy minimisation (REM). Initially, REM consisted of a 50 stepsteepest descent followed by 100 step conjugate gradient minimisation.RMD was performed by simulated heating of the structures to 300K for 1ps, followed by 500K for 1 ps. The temperature was gradually lowered to300K over 2 ps and finally for 2 ps at 200K. REM was performed againwith a 50 step steepest descent, 200 step conjugate gradient followed bya 300 step Powell minimisation. The final structures were examined toobtain a mean pairwise rms difference over the backbone heavy atoms (N,Cα and C). Twenty of the 50 structures had a mean rmsd<0.5 Å for allbackbone atoms (O, N, C).

Molecular Modelling

A model of cycle 12, shown in FIG. 7, was created from the NMR structureof 7 by deleting all NMR constraints, fusing the ornithine side chainamine to the C-terminal carboxylate of d-Arg to form an amide, andminimising using Powell forcefield (1000 iterations). The modelledstructure was then superimposed on the NMR structure with an rmsd 0.224Å.

Receptor-Binding Assay

Assays were performed with fresh human PMNs, isolated as previouslydescribed (Sanderson et al, 1995), using a buffer of 50 mm HEPES, 1 mMCaCl₂, 5 mM MgCl₂, 0.5% bovine serum albumin, 0.1% bacitracin and 100 μMphenylmethylsulfonyl fluoride (PMSF). In assays performed at 4° C.,buffer, unlabelled human recombinant C5a (Sigma) or peptide,Hunter/Bolton labelled ¹²⁵, —C5a (˜20 pM) (New England Nuclear, MA) andPMNs (0.2×10⁶) were added sequentially to a Millipore Multiscreen assayplate (HV 0.45) having a final volume of 200 μL/well. After incubationfor 60 min at 4° C., the samples were filtered and the plate washed oncewith buffer. Filters were dried, punched and counted in an LKB gammacounter. Non-specific binding was assessed by the inclusion of 1 mMpeptide or 100 nM C5a which typically resulted in 10-15% total binding.

Data was analysed using non-linear regression and statistics withDunnett post test.

Myeloperoxidase Release

Cells were isolated as previously described (Sanderson et al, 1995) andincubated with cytochalasin B (5 μg/mL, 15 min, 37° C.). Hank's BalancedSalt solution containing 0.15% gelatin and peptide was added on to a 96well plate (total volume 100 μL/well), followed by 25 μL cells(4×10⁶/mL). To assess the capacity of each peptide to antagonise C5a,cells were incubated for 5 min at 37° C. with each peptide, followed byaddition of C5a (100 nM) and further incubation for 5 min. Then 50 μL ofsodium phosphate (0.1M, pH 6.8) was added to each well, the plate wascooled to room temperature, and 25 μL of a fresh mixture of equalvolumes of dimethoxybenzidine (5.7 mg/mL) and H₂O₂ (0.51%) was added toeach well. The reaction was stopped at 10 min by addition of 2% sodiumazide. Absorbances were measured at 450 nm in a Bioscan 450 platereader, corrected for control values (no peptide), and analysed bynon-linear regression.

In Vivo Assays of Anti-Inflammatory Activity

The following well-known in vivo assay systems may be used to assess theanti-inflammatory activity of compounds of the invention. All assay dataare analysed using non-linear regression analysis and Student's t-test,analysis of variance, with p<0.05 as the threshold level ofsignificance.

(a) Carrageenan Paw Oedema

Anaesthetised (i.p. ketamine & xylazine) Wistar rats (150-200 g) or micewere injected with sterilised air (20 ml day 1, 10 ml day 4) into thesubcutaneous tissue of the back. The cavity can be used after 6 days,whereupon carrageenan (2 ml, 1% w/w in 0.9% saline) was injected intothe air pouch and exudate was collected after 10 hr. Test compounds areadministered daily after Day 6 and their anti-inflammatory effectsassayed by differential counting of cells in the air-pouch exudate.Animals were killed at appropriate times after injection and 2 ml 0.9%saline was used to lavage the cavity, lavage fluids were transferred toheparinised tube and cells were counted with a haemocytometer andDiff-Quik strained cytocentrifuged preparation.

Alternatively, a routine carrageenan paw oedema was developed in Wistarrats by administering a pedal injection of carrageenan to elicit oedemawhich is visible in 2 h and maximised in 4 h. Test compounds are given40 min before inflammagen and evaluated by microcaliper measurements ofpaws after 2 & 4 hr. See Fairlie, D. P. et al (1987). Also see Walkerand Whitehouse (1978).

(b) Adjuvant Arthritis.

Adjuvant arthritis was induced in rats (3 strains) either microbially(injection of heat-killed Mycobacterium tuberculosis) or chemically(with pyridine) by inoculation with the arthritogenic adjuvantco-administered with oily vehicles (Freund's adjuvants) in the tailbase. (See Whitehouse, M. W., Handbook of Animal Models for theRheumatic Diseases, Eds. Greenwald, R. A.; Diamond, H. S.; Vol. 1, pp.3-16, CRC Press).

Within 13 days the adjuvant arthritis is manifested by localinflammation and ulceration in the tail, gross swelling of all fourpaws, inflammatory lesions in paws and ears, weight loss and fever.These symptoms, which are similar to those of inflammatory disease inhumans (Winter and Nuss, 1966), can be alleviated by agents such asindomethacin or cyclosporin which also show beneficial effects in man(eg. Ward and Cloud, 1966). Without drug treatment at Day 14, arthriticrats had hypertrophy of the paws, reduced albumin but raised acute phasereaction proteins in serum, and depressed hepatic metabolism ofxenobiotics as indicated by prolonged barbiturate-induced sleepingtimes.

To assess activity, compounds were administered for 4 days orally (<10mg/kg/day) or i.p. from Days 10-13 following inoculation witharthritogen (Day 0). The inflammation was either not visible or verysignificantly reduced in rear or front paws as assessed by microcalipermeasurements of paw thickness and tail volume, as well as by grossinspection of inflammatory lesions. Animals are sacrificed by cervicaldislocation on Day 18 unless arthritis signs are absent, whereuponduration of observations is continued with special permission from theEthics committees. Experiments are staggered to maximise throughput andallow early comparisons between compounds. This routine assay iswell-accepted as identifying anti-inflammatory agents for use in humans.

EXAMPLE 1 Structure-Activity Relationship of C5a Agonists

We have focused on the C-terminal residues of C5a, in order to explorestructure-activity relationships in the search for peptide sequenceswith potent agonist activity. Many of these peptides are full agonistsrelative to C5a, but have markedly lower potency (Sanderson et al, 1994,1995; Finch et al, 1997). Our initial structure-activity investigationshave been particularly informative. Mutating the decapeptide C-terminusof C5a (SEQ ID NO: 1, C5a₆₅₋₇₄, ISHKDMQLGR) twice with I₆₅Y and H₆₇F(eg. 2) led to enhancement of agonist potency by about 2 orders ofmagnitude. These results are summarised in Table 2. Analyses ofRamschandran plots and 2D NMR spectra for compound 2 suggested thatcertain structural features, namely a twisted “helix-like” backboneconformation for residues 65-69 and a β-turn for residues 71-74, mightbe responsible for activity. These preliminary results provided someinsight to structural requirements for tight binding to a C5a receptor.

TABLE 2 Pharmacological Activity of C5a Agonist Analogues* PMN FetalEnzyme Binding Peptide Artery Release Affinity No. Peptide EC₅₀ (μM)EC₅₀ (μM) IC₅₀ (μM) SEQ. C5a₆₅₋₇₄ >1000 >1000 >1000 ID NO: 1(ISHKDMQLGR) SEQ. YSFKDMQLGR 9.6 92 1.3 ID NO: 2 SEQ. YSFKDMPLaR 0.5 723.7 ID NO: 3 SEQ. YSFKPMPLaR 0.2 4.3 6.0 ID NO: 4 SEQ. C5a₃₇₋₄₆- 0.065.9 0.7 ID NO: 5 ahxYSFKPMPLaR SEQ. C5a₁₂₋₂₀- 0.08 0.7 0.07 ID NO: 6ahxYSFKPMPLaR C5a 0.02 0.03 0.0005 *Finch et al, 1997

Compounds 4, 5 and 6 in Table 2 are the highest affinity small C5aagonists so far known, with up to 25% C5a potency in human fetal artery,5% C5potency in human PMN enzyme release assays and 1% C5a affinity forPMN C5aR (Finch et al., 1997). For the PMN receptor, these compoundshave up to 100-fold higher apparent affinity than any small moleculepreviously described in the literature.

The “high” affinities (70 nM-6 μM) of these agonist analogues for C5aRin intact PMN cells have enabled us to identify a common topographicalfeature in peptide agonists that correlates with expression ofspasmogenic activities and enzyme-release assays in human PMNs. Thispreferred backbone conformation is a type II β-turn.

The small size of these agonist peptides makes them amenable tosynthetic modification to optimise their affinities, activities, andbioavailabilities, and hence useful as mechanistic probes of receptoractivation.

EXAMPLE 2 NMR Structure of C5a Antagonist

We used two dimensional nuclear magnetic resonance spectroscopy todetermine the three dimensional structure of 7 and found that whilethere is no discernible structure in water, there is evidence of astable gamma-turn structure in dimethylsulfoxide.

The 1D ¹H-NMR spectrum of peptide 7 in d₆-DMSO at 24° C. shows 4distinct resonances for amide-NH protons, as summarized in Table 3. Toestablish their possible involvement in intramolecular hydrogen bonds, adeuterium exchange experiment was performed by adding a 10-fold excessof D₂O to the solution. Two of the amide-NH doublets disappearedimmediately, along with resonances attributable to the N-terminalmethylamine protons. However, the other two amide NH resonances, as wellas a broad resonance at approximately 8.05 ppm, persisted for up to 6.5hours (FIG. 2). These three slowly-exchanging protons are assigned tothe amide NHs of Trp and d-Cha and the side chain amine of Lys, the slowexchange behaviour being characteristic of hydrogen-bonding. The amineassignment was established from the TOCSY spectrum where cross peakswere observed between the protonated amine and the ε, δ and γ CH₂protons. A temperature dependence study (20-60° C.) of the amide-NHchemical shifts (Δδ/T=2.5 ppb/deg, dCha-NH; 6 ppb/deg, Trp-NH; 6.5 ppb,Lys-NH; 8.7 ppb, Arg-NH) unambiguously confirmed the involvement of thedCha-NH only in intramolecular hydrogen bonding.

TABLE 3 ¹H-NMR Assignments^(a) for 7 in d₆-DMSO Residue ^(b)HN Hα Hβ HγOthers MePhe — 4.06 3.09, — ^(c)7.17, 7.29; ^(d)2.46; ^(f)8.98 3.06 Lys8.83 4.54 1.74, 1.32 ^(e)1.51; ^(f)2.74, ^(g)7.76 (NH₂) 1.55 Pro — 4.302.084,  1.88, ^(e)3.61, ^(f)3.48 1.74 1.78 d-Cha 7.91 4.35 1.19, 0.76^(e)1.43, 1.08; ^(f)1.61, 1.58; 1.06 0.73 Trp 8.01 4.65 3.11, —^(c)6.97, 7.06, 7.13, 7.32, 2.94 7.65; ^(g)10.80 d-Arg 8.44 4.20 1.73,1.42 ^(e)3.08; ^(g)7.60 1.58 ^(a)Referenced to residual d₆-DMSO at 2.50ppm. ^(b)Amide NHS, ^(3′)NH-CaH values (Hz): 7.91 (Lys), 7.77 (d-Arg),8.34 (Trp), 8.53 (d-Cha). ^(c)Aromatics ^(d)N-Me. ^(e)Hδ. ^(f)Hε^(g)NH/NH₂ amine.

A series of 2D ¹H-NMR spectra were measured for 7 at 24° C. in d₆-DMSOto determine the three-dimensional structure. TOCSY and DFQ-COSYexperiments were used to identify residue types, while sequentialassignments were made from analysis of NOESY data. From a series of 100structures generated from NOESY data, fifty of the lowest energystructures were subjected to restrained molecular dynamics (200K-500K)and energy minimised. A set of 20 calculated structures with a root meansquare deviation (rmsd)<0.5 Å (backbone atoms) are superimposed in FIG.3, and clearly depict a turn conformation.

In combination, the NMR constraint data, ³NH-CαH values, deuteriumexchange and temperature dependence data establish an unusual turnstructure for hexapeptide 7 which is constrained by up to three hydrogenbond, as shown in FIG. 4. The evidence is very strong for oneintramolecular hydrogen bond from dCha-NH . . . OC-Lys (2.72 Å, N—H . .. O angle 157°, C═O . . . H angle 84°), forming a 7-membered ring thatdefines an inverse γ-turn. The dChaNH—O-TrpNH angle is 56.4°. Thedeuterium exchange data and NMR constraint data together point to asecond intramolecular hydrogen bond Trp-NH . . . OC-Lys (3.31 Å, N—H . .. O angle 159°, CO . . . H angle 137.3°) forming a 10-membered ringcharacteristic of a β-turn. The φ and ψ angles (100 ₂=−58.4°, ψ₂=62.0°;φ=96.6°, ψ₃=16.6°) most closely match a type II β-turn (Bandekar, 1993;Hutchinson and Thornton, 1994) which is distorted by the presence of theγ-turn wholly within the β-turn.

To our knowledge this is the first example of an intramolecular hydrogenbond between residues within a β-turn, although there are many examplesof hydrogen bonds between a residue within the “10 membered ring” of aβ-turn and a residue outside of it (Bandekar, 1993). A third hydrogenbond (2.76 Å, N—H . . . O angle 160.3°), between the side-chain amine ofLys and the C-terminal carboxylate, is suggested by the NMR constraintdata, by slow NH/ND exchange and by detection of a weak NOE betweenLys-NH . . . Trp-αCH₂. This may further constrain the molecule into theobserved turn conformation. Such ion-pairing is common in dipolaraprotic solvents such as dimethylsulphoxide and may also be relevant ina hydrophobic protein environment.

NMR solution structures have also been determined for several of thecyclic antagonists described in the following examples, and show that ineach case the type II β-turn is preserved and stabilized by the cyclicstructure.

The constraining β and γ turns proposed in the linear peptide 7 haveparallels in cyclic peptides. We have previously detected overlapping βand γ turns in a cyclic octapeptide from ascidiacyclamide (Abbenante etal, 1996). Combinations of a β- and γ-turn have also been found in thebackbones of cyclic penta- and hexapeptides, particularly thosecontaining alternating D- and L-amino acids (Marraud and Aubry 1996;Fairlie et al, 1995; Kessler et al, 1995; Stradley et al, 1990). Forexample a type II β-turn and an inverse γ-turn have been identified incyclic antagonists c-(D-Glu-Ala-D-allo-Ile-Leu-D-Trp] (Ihara et al,1991; Coles et al, 1993; Ihara et al, 1992; Bean et al, 1994) andc-(D-Asp-Pro-D-Val-Leu-D-Trp) (Bean et al, 1994) for endothelinreceptors, and in members of the rhodopsin family of G protein-coupledreceptors with seven transmembrane domains (X. -M. Cheng et al, 1994).In the latter case, as in 7, an inverse γ-turn forms between residues(Asp-CO . . . Val-NH, Lys-CO . . . dCha-NH) that flank the proline.

EXAMPLE 3 Structure-Activity Relationships in Vitro

We also examined the receptor-binding and antagonist activity of thehexapeptide 7 for comparison with our compounds. The previous-report byKonteatis et al (1994) concerned the ability of 7 to compete with C5abinding to receptors on isolated PMN membranes (IC₅₀ 70 nM), which isnot necessarily physiologically relevant. We examined competitionbetween 7 and C5a using intact PMN cells, and found that, under theseconditions, 7 binds with much lower receptor affinity of IC₅₀ 1.8 μM. Weconfirmed that 7 is a full antagonist with no agonist properties. Theseresults are summarized in FIG. 5a and Table 4. The relative affinity(ratio) of 7 for the C5aR in intact PMNs in our assays was similar tothat previously reported for isolated PMN membranes.

We have also found that 7 shows antagonist activity against both C5a(FIG. 5b) and a C-terminal agonist decapeptide analogue 4 (YSFKPMPLaR)(Finch et al, 1997) of the C-terminus C5a₆₅₋₇₄, suggesting that it actson site 2 of the receptor. Compounds 7 and 4 have similar μM affinityfor the receptor C5aR on intact polymorphonuclear leukocytes, as shownin Table 4.

A new discovery from the data in Table 4 is the linear correlationbetween the log of binding affinities and the log of antagonistpotencies for these Site 2 antagonists (compounds 7-12, Table 4). Theimportance of this linear relationship is that since receptor affinityand antagonist activity are directly proportional, the experimentallysimpler approach of measuring receptor binding may be used to estimatethe antagonist activity for such small compounds, provided that there isno evidence of agonist activity.

TABLE 4 Receptor-Binding Affinities^(a) and Antagonist Activities^(b) inHuman PMNs Receptor Affinity^(a) Antagonist Potency^(b) Agonist CompoundIC₅₀ (μM) IC₅₀ (μM) Activity^(c) SEQ. ID NO: 7 MeFKP (dCha) Wr 1.8 (15)0.085 (9) No SEQ. ID NO: 8 MeFKP (dCha) Wr-CONH₂ 14 (5) 0.5 (3) No SEQ.ID NO: 9 MeFKP (dCha) WR 11 (5) 0.7 (3) No SEQ. ID NO: 10 MeFKPLWR 144(1) >1000 (3) nd SEQ. ID NO: 11 Ac-F-[KP (dCha) Wr 3.2 (40) 0.090 (5) NoSEQ. ID NO: 12 Ac-F-[OP (dCha) Wr 0.28 (6) 0.012 (4) No SEQ. ID NO: 4YSFKPMPLaR 6.0^(d) — Yes SEQ. ID NO: 1 C5a₆₅₋₇₄, ISHKDMQLGR >1000^(e) —— C5a 0.0008 (9) — Yes Number of experiments in parenthesis. Correctedfor amino acid content Square brackets indicate cyclic portion. nd = notdetermined ^(a)50% reduction in binding of ¹²⁵I-C5a to intact human PMNs^(b)50% reduction in myeloperoxidase secretion from human PMNs mediatedby 100 nM C5a ^(c)Agonist activity in dose range 0.1 nM-1 nM ^(d)Finchet al, 1997; ^(e)Kawai et al, 1991

It has previously been proposed that the C-terminus of C5a and ofagonist peptides is essential for activity, due to its interaction witha positively-charged Arg206 of the receptor (DeMartino et al, 1995). Weconfirm here that the C-terminal carboxylate is indeed important foractivity (8 vs. 7), but wondered whether the origin of this effect mightbe due to hydrogen bonding between the carboxylate anion and thepositively charged amine side chain of Lys. Conversion to the amide (8)certainly reduces both receptor-affinity and antagonist activityapproximately 5-fold. Changing chirality of the Arg-Cα (9 vs. 7) causesa similar reduction in activity, and replacing dCha with the less bulkyLeu residue (10) is also detrimental to receptor binding. However,potency is recovered for cyclic compounds 11 and 12, in which an amidebond is tolerated at the C-terminus, consistent with the structuralinterpretation above that the advantage of the carboxylate in 7 may beassociated with intramolecular hydrogen bonding. The replacement of thishydrogen bond it 7 with a covalent amide bond in 11 and 12 moreeffectively stabilizes the turn conformation.

FIG. 5C compares C5aR binding and antagonist potency in vitro on humanPMNs for compounds 15 and 17 with those for compound 7. Both 15 and 17are potent inhibitors at nM concentrations of the action of C5a and thebinding of ^(125I-C)5a to its receptor (e.g. 4, Kb=1.4 nM). Their cyclicnature and the acetylation at the N-terminal phenylalanine both protectagainst the proteolytic degradation typically encountered by peptides,making such cyclic compounds more suitable than acyclic peptides as drugcandidates. The results are shown in Table 5.

TABLE 5 Receptor Binding and Antagonist Activity of Cyclic Molecules

Receptor Affinity Agonist Compound n R Isomer* μM Activity SEQ. ID NO:13 1 H S- 9 No SEQ. ID NO: 14 R- 34 No SEQ. ID NO: 15 2 H S- 0.3 No SEQ.ID NO: 16 R- 3.7 No SEQ. ID NO: 17 3 Ac S- 0.3 No SEQ. ID NO: 11 Ac R-38 No SEQ. ID NO: 18 4 Ac S- 3.2 No SEQ. ID NO: 12 Ac R- 51 No Refers tostereochemistry of Arg side chain

EXAMPLE 4 Cyclic Antagonists of C5a

Some examples of these cyclic antagonists and their apparentreceptor-binding affinities and antagonist potencies are given in Tables4, 5 and 6 as well as in FIGS. 5 and 6. In the tables the single lettercode for amino acids is used.

TABLE 6 PEPTIDE pD₂ ± SE^(a) IC₅₀ (M)^(a) (n) pD₂ ± SE^(b) IC₅₀ (M)^(b)(n) Effect of Cyclisation on Antagonist Binding Affinity and AntagonistPotency SEQ. ID NO: 11 AcF-[KPdChaWr] 5.49 ± 0.22 3.2 4 7.07 ± 0.29 0.095 SEQ. ID NO: 12 AcF-[OPdChaWr]  6.44 ± 0.14* 0.4 9 7.30 ± 0.09 0.05 9SEQ. ID NO: 19 [FWPdChaWr]  4.37 ± 0.36* 43 3 nd SEQ. ID NO: 20AcF-[KMdChaWr] 4.81 ± 0.06 15 2 nd SEQ. ID NO: 21 AcF-[KKdChaWr] 3.94 ±0.4  116 3 4.88 13 1 Effect of length of linker in cycle on antagonistbinding affinity and antagonist potency SEQ. ID NO: 22 AcF-[XPdChaWr]5.02 ± 0.07 9.5 3 4.71 ± 0.23 20 3 SEQ. ID NO: 23 AcF-[X²PdChaWr]  4.77± 0.14* 17 3  6.09 ± 0.08* 0.8 3 SEQ. ID NO: 12 AcF-[OPdChaWr]  4.60 ±0.06* 16 4 6.42 ± 0.10 0.4 4 SEQ. ID NO: 24 AcKF-[OPdChaWr] 4.96 ± 0.0311 3 6.73 0.2 1 SEQ. ID NO: 14 F-[XPdChaWr]  4.39 ± 0.10* 41 3 nd SEQ.ID NO: 16 F-[X²PdChaWr] 5.42 ± 0.05 3.8 3 6.70 ± 0.04 0.4 3 SEQ. ID NO:25 F-[OPdChaWr] 5.51 ± 0.07 3.1 3  5.79 ± 0.34* 1.6 3 SEQ. ID NO: 26F-[KPdChaWr] 5.09 ± 0.08 8.1 3  5.55 ± 0.57* 2.8 3 Effect of L-Arg onantagonist binding affinity and antagonist potency SEQ. ID NO: 17AcF-[OPdChaWr] 6.57 ± 0.05 0.3 3  7.91 ± 0.17* 0.01 3 SEQ. ID NO: 13F-[XPdChaWr] 4.98 ± 0.05 10 3  5.63 ± 0.13* 2.4 3 SEQ. ID NO: 15F-[X²PdChaWr]  6.50 ± 0.04* 0.3 5 7.36 ± 0.13 0.04 3 SEQ. ID NO: 27F-[OPdChaWr]  7.21 ± 0.01* 0.06 3 7.41 ± 0.14 0.04 3 SEQ. ID NO: 28F-[KPdChaWr]  6.50 ± 0.12* 0.3 4 6.69 ± 0.04 0.2 3

These results demonstrate:

-   -   (1) that the cyclic molecules have higher apparent receptor        affinity and may be more potent antagonists than acyclic        (linear) peptides,    -   (2) that one of the two possible cyclic diastereomers is        consistently favoured for binding to the C5a receptor, and it is        surprisingly the opposite stereochemistry (L-arginine) to that        favoured in the linear compounds (D-arginine)    -   (3) that the cycles have an optimum ring size for        receptor-binding,    -   (4) that there is a pseudo-linear relationship between log        (antagonist potency) and log (receptor affinity).

Tables 5 and 6 list the C5a receptor affinities of some examples ofcyclic antagonists of C5a, and their ability to bind to, and inhibit,binding of C5a to human PMNs is illustrated in FIG. 6. Surprisinglythese data show that the L-arginine is preferred over the D-arginine, incontrast to the linear compound 7 in which the D-arginine confers higheraffinity for the receptor than does L-arginine. The data also show thatthe size of the macrocycle is optimal when n=2 or 3, the smaller cyclewhere n=1 and the larger cycle when n=4 being clearly less active. Thisrequirement for a tightly constrained cycle is probably due to the needto correctly position the attached side chain residues of, for example,Trp, dCha, Arg and Phe for interaction with the receptor.

EXAMPLE 5 Computer Modelling of Antagonist Structures

FIG. 7 compares the computer-modelled structure of the cyclic antagonist12 with the NMR solution structure for the acyclic antagonist 7. Thesebackbone structures are strikingly similar, and strongly suggest thatthe receptor-binding conformations of these molecules involve the sameturn structure. Compound 12, a more potent antagonist than 11, also hasa shorter linker, which tightens the turn and slightly alters theconformational space accessible to the key side chains of Phe, dCha, Trpand Arg. The conformational limitations placed on the hexapeptidederivative 12 by the cycle are responsible for a≧10⁴ increase inreceptor-binding affinity over the conformationally flexible decapeptideC-terminus of C5a (1, Table 2).

There is a correlation between binding affinities and antagonist potencyfor the site 2 antagonists (compounds 7-12, Table 2). It thus appearsthat antagonist potency is dependent upon changes that occur at site 2alone. Without wishing to be bound by any proposed mechanism, we believethat this may be because the mechanism of antagonism is related toconformational change to a turn conformation induced by 7 at site 2 ofthe receptor.

EXAMPLE 6 Characterisation of C5aRS on Different Cells

Currently there is no information about different types of C5aRs. Wehave previously shown marked differences in the responsiveness ofdifferent cells containing functional C5aRs to agonists (Sanderson etal, 1994, 1995; Finch et al, 1997) and we can now provide moreinformation by examining potency and efficacy of selective agonists andantagonists relative to human recombinant C5a. For agonists, the tissueor cell selectivity may reveal functionally different receptors. Bindingassays using human PMNs, U937 cells, or circulating monocytes are usedto determine affinities for C5aRs. Selectivity for different C5aRs isascertained by differential antagonism. This combined approach allowspharmacological characterisation of new agonists or antagonists, and maylead to a potential functional classification of C5aRson differentcells.

EXAMPLE 7(a) Neutropenia and C5a Antagonism In Vivo

Compounds were evaluated in an acute model of C5a-induced neutropenia.Transient neutropenia maximises 5 min after i.v. C5a and is profound,with >90% of circulating neutrophils disappearing from circulation ateffective doses of C5a, as shown in FIG. 8. The neutropenia is due totransient adherence of circulating neutrophils to the vascularendothelium. Preliminary data show that neutropenia caused by i.v. C5ais blocked by a C5a antagonist. For example, F-[OPdChaWR], (1 mg/kg),given prior to 2 μg C5a i.v., inhibits C5a-induced neutropenia in vivo(FIG. 8)

EXAMPLE 7(b) Inhibition of Lipopolysaccharide-Induced Effects by C5aAntagonists

LPS causes rapid neutropenia in rats. If this effect of LPS is blockedby C5a antagonists, then C5a may be of major importance in the acuteeffects of LPS, and the results shown in FIG. 9 were in agreement withthis hypothesis. C5a antagonists were injected (bolus i.v.) 10 min priorto challenge with LPS. Rats were anaesthetised, and blood samples (0.3ml) were taken for measurements of PMNs. PMNs are isolated andquantified. Preliminary results show that F-[OPdChaWR], (1 mg/kg), givenprior to i.v. LPS, inhibits neutropenia.

The results also indicate that the C5a antagonist inhibits the increasein haematocrit caused by LPS, showing that vascular leakage of serumcaused by LPS is also inhibited.

These results demonstrate that C5a receptor antagonists, such as thosedescribed in this invention, may have therapeutic utility in septicaemicindividuals. The ability to inhibit the adherence of PMNs to vascularendothelium, and to inhibit the vascular leakage to LPS as shown by thereduction of haematocrit values, indicates powerful anti-inflammatoryeffects of these compounds against proinflammatory stimuli activatingthe complement system, such as endotoxin or LPS.

EXAMPLE 8 In Vivo Activity of Cyclic C5a Antagonists

Preliminary experiments in rats have revealed that the cyclicantagonists summarized in Table 5 are active at less than 20 mg/kg asanti-inflammatory agents in suppressing the onset of eithercarrageenan-induced paw oedema or adjuvant-induced polyarthritis. Themaximally effective dosages for even moderately-effective antagonistsare 10 mg/kg or less, given i.p. or p.o. Many anti-inflammatory drugscurrently used in humans were initially evaluated in such assays, andalso showed activity in these rat models of inflammation. Thesepreliminary indications of efficacy in vivo indicate that C5aantagonists have therapeutic potential in human inflammatory conditions.

Using the rat carrageenan paw oedema assay, we found that a compound,AcF-[O—P-dCha-W-r], which is 100 times less active than 17 in vitro as aC5a antagonist in PMNs, has some in vivo activity in rats given 1 mg/kgof the compound I.P, 30 min prior to the carageenan injection. Pawswelling was measured for up to 4.5 hr. The results, shown in FIG. 10,suggest that even this weak C5a antagonist significantly inhibitsdevelopment of the oedema after 180 and 270 min. This anti-inflammatoryactivity suggests that C5a receptor antagonists, such as those describedin this invention, may have therapeutic activity in diseases involvingvascular leakage following inflammatory stimuli.

In recent years there have been many attempts to mimic β- and γ-turnpeptides that represent bioactive protein surfaces, resulting in notablemimetics for RGD (arginine-glycine-aspartate) peptides, somatostatin andopioid peptides, to name a few derived through structure-activityrelationships (see for example Marraud and Aubry, 1996; Fairlie et al,1995). Most of these examples preserve a turn structure throughcyclisation of the peptide. On the other hand, there are comparativelyfew short acyclic peptides that have been found to have substantial turnstructure in solution (Dyson et al, 1988; Rizo and Gierasch, 1992;Pràcheur et al, 1994). It is usually argued that short acyclic peptidesadopt a myriad of solution structures that may include small populationsof turn structures that are responsible for bioactivity.

This invention describes a series of conformationally-constrainedturn-containing molecules that are preorganized for binding to the sameG protein-coupled receptor(s) of human cells that are targeted by humanC5a. The invention is applicable to other C protein-coupled receptors.

The principal feature of the compounds of the invention is thepreorganized arrangement, which brings at least three hydrophobic groupsand a charged group into neighbouring space, creating a hydrophobicsurface ‘patch’. These results enable the design and development of evenmore potent conformationally-constrained, small molecule antagonists ofC5a.

In the light of the aforementioned prior art, it was surprising to findthat a C-terminal carboxylate was not necessary in our compounds inorder to obtain good receptor-binding or antagonist activity. The cyclicantagonists have an amide bond at the ‘C-terminal’ arginine position.The replacement of the carboxylate in 7 with a covalent amide bondeffectively stabilises the required turn conformation.

Cyclic and non-peptidic antagonists have several important advantagesover peptides as drugs. The cycles described in this invention arestable to proteolytic degradation for at least several hours at 37° C.in human blood or plasma, or in human or rat gastric juices or in thepresence of digestive enzymes such as pepsin, trypsin and chymotrypsin.In contrast, short peptides composed of L-amino acids are rapidlydegraded to their component amino acids within a few minutes under theseconditions. A second advantage lies in the constrained singleconformations adopted by the cyclic and non-peptidic molecules, whereasacyclic or linear peptides are flexible enough to adopt severalstructures in solution other than the required receptor-bindingstructure. Thirdly, cyclic and non-peptidic compounds such as thosedescribed in this invention are usually more lipid-soluble and morepharmacologically bioavailable as drugs than peptides, which can rarelybe administered orally. Fourthly, the plasma half-lives of cyclic andnon-peptidic molecules are usually longer than those of peptides.

It will be apparent to the person skilled in the art that while theinvention has been described in some detail for the purposes of clarityand understanding, various modifications and alterations to theembodiments and methods described herein may be made without departingfrom the scope of the inventive concept disclosed in this specification.

References cited herein are listed on the following pages, and areincorporated herein by this reference.

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1. A compound, which has antagonist activity against a C5a receptor, hasno agonist activity against a C5a receptor, and has the general formulaII:

where A is H, alkyl, aryl, NH₂, NHalkyl, N(alkyl)₂, NHaryl or NHacyl; Bis an alkyl, aryl, phenyl, benzyl, naphthyl or indole group, or the sidechain of a D- or L-amino acid selected from the group consisting ofphenylalanine, homophenylalanine, tryptophan, homotryptophan, tyrosine,and homotyrosine; C is the side chain of D-, L- or homo-amino acidselected from the group consisting of proline, alanine, leucine valine,isoleucine, arginine, histidine, aspartate, glutamate, glutamine,asparagine, lysine, tyrosine, phenylalanine, cyclohexylalanine,norleucine, tryptophan, cysteine and methionine; D is the side chain ofa D- or L-amino acid selected from the group consisting ofcyclohexylalanine, homocyclohexylalanine, leucine, norleucine,homoleucine, homonorleucine and tryptophan; E is the side chain of a D-or L-amino acid selected from the group consisting of tryptophan andhomotryptophan; F is the side chain of a D- or L-amino acid selectedfrom the group consisting of arginine, homoarginine, lysine andhomolysine or is one of the following side-chains

or another mimetic of an arginine side chain, where X is NCN, NNO₂,CHNO₂ or NSO₂NH₂; n is an integer from 1 to 4, and R¹ is H or an alkyl,aryl, CN, NH₂, OH, —CO—CH₂CH₃, —CO—CH₃, —CO—CH₂CH₂CH₃, —CO—CH₂Ph, or—CO-Ph; and X¹ is —(CH₂)_(n)NH— or (CH₂)_(n)—S—, —(CH₂)₂O—, —(CH₂)₃O—,—(CH₂)₃—, —(CH₂)₄—, or —CH₂COCHRNH—, where R is the side chain of anycommon or uncommon amino acid, and where n is an integer of from 1 to 4.2. The compound according to claim 1, which is a compound selected fromthe group consisting SEQ ID NOS: 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27 and
 28. 3. The compound according to claim 1,in which n is 2 or
 3. 4. The compound according to claim 1, in which Fis one of the following side-chains

or another mimetic of an arginine side chain; where X is NCN, NNO₂,CHNO₂ or NSO₂NH₂; n is an integer from 1 to 4, and R¹ is H or an alkyl,aryl, CN, NH₂, OH, —CO—CH₂CH₃, —CO—CH₃, —CO—CH₂CH₂CH₃, —CO—CH₂Ph, or—CO-Ph; B is an indole, indole methyl, benzyl, phenyl, naphthyl,naphthyl methyl, cinnamyl group, or any other derivative of the aromaticgroup; and C is D- or L-cyclohexylalanine (Cha), leucine, valine,isoleucine, phenylalanine, tryptophan or methionine.
 5. The compoundaccording to claim 1, which has the formulaAc-Phe-[Lys-Pro-(dCha)-Trp-Arg] orAc-Phe-[Orn-Pro-(dCha)-Trp-Arg].
 6. The compound according to claim 1,in which A is L-arginine.
 7. The compound according to claim 1, in whichF is a L-amino acid.
 8. The compound according to claim 7, in which F isL-arginine.
 9. A composition comprising a compound according to claim 1,together with a pharmaceutically-acceptable carrier or excipient. 10.The composition according to claim 9, wherein in the compound of formulaII, F is L-arginine.
 11. The composition according to claim 9, whereinthe compound of formula II is a compound selected from the groupconsisting SEQ ID NOS: 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27 and
 28. 12. The composition according to claim 9,wherein the compound of formula II has the formulaAc-Phe-[Lys-Pro-(dCha)-Trp-Arg] orAc-Phe-[Orn-Pro-(dCha)-Trp-Arg].
 13. The composition according to claim9, in which F is L-arginine.
 14. The composition of claim 9, wherein inthe compound of formula II, F is a L-amino acid.
 15. The compoundaccording to claim 14, in which R¹ is methyl, ethyl, propyl, or butyl.16. The composition according to claim 9, wherein in the compound offormula II F is one of the following side-chains

or another mimetic of an arginine side chain; where X is NCN, NNO₂,CHNO₂ or NSO₂NH₂; n is an integer from 1 to 4, and R¹ is H or an alkyl,aryl, CN, NH₂, OH, —CO—CH₂CH₃, —CO—CH₃, —CO—CH₂CH₂CH₃, —CO—CH₂Ph, or—CO-Ph; B is an indole, indole methyl, benzyl, phenyl, naphthyl,naphthyl methyl, cinnamyl group, or any other derivative of the aromaticgroup; and C is D- or L-cyclohexylalanine (Cha), leucine, valine,isoleucine, phenylalanine, tryptophan or methionine.
 17. The compositionaccording to claim 16, wherein in the compound of formula II, R¹ ismethyl, ethyl, propyl, or butyl.
 18. A method of antagonizing theactivity of a C5a receptor on a cell, comprising contacting the cellwith the compound of claim 1 in an amount sufficient to antagonize theactivity of the C5a receptor on the cell.
 19. The method according toclaim 18, wherein in the compound of formula II, F is L-arginine. 20.The method according to claim 18, wherein the compound of formula II isa compound selected from the group consisting SEQ ID NOS: 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 and
 28. 21. Themethod according to claim 18, wherein the compound of formula II has theformulaAc-Phe-[Lys-Pro-(dCha)-Trp-Arg] orAc-Phe-[Orn-Pro-(dCha)-Trp-Arg].
 22. The method according to claim 18,wherein in the compound of formula II, F is L-arginine.
 23. The methodof claim 18, wherein the cell is in a mammal and said contactingcomprises administering the compound to said mammal.
 24. The methodaccording to claim 18, wherein in the compound of formula II, F is aL-amino acid.
 25. The method of claim 24, wherein said mammal is ahuman.
 26. The method according to claim 18, wherein in the compound offormula II F is one of the following side-chains

or another mimetic of an arginine side chain; where X is NCN, NNO₂,CHNO₂ or NSO₂NH₂; n is an integer from 1 to 4, and R¹ is H or an alkyl,aryl, CN, NH₂, OH, —CO—CH₂CH₃, —CO—CH₃, —CO—CH₂CH₂CH₃, —CO—CH₂Ph, or—CO-Ph; B is an indole, indole methyl, benzyl, phenyl, naphthyl,naphthyl methyl, cinnamyl group, or any other derivative of the aromaticgroup; and C is D- or L-cyclohexylalanine (Cha), leucine, valine,isoleucine, phenylalanine, tryptophan or methionine.
 27. The methodaccording to claim 26, wherein in the compound of formula II, R¹ ismethyl, ethyl, propyl, or butyl.
 28. A method of treating inflammatoryarthritis mediated by a C5a receptor, comprising the step ofadministering an effective amount of a compound according to claim 10 toa mammal in need thereof.
 29. The method of claim 28, wherein the mammalis a human.
 30. The method of claim 28, wherein the inflammatoryarthritis is rheumatoid arthritis.
 31. A method of treating inflammatoryarthritis, comprising the step of administering an effective amount of acompound according to claim 1 to a mammal in need thereof.
 32. Themethod of claim 31, wherein the mammal is a human.
 33. The method ofclaim 31, wherein the inflammatory arthritis is rheumatoid arthritis.34. A compound which is an antagonist of the C5a receptor, and has theformula IV:

where A is any common or uncommon, basic, charged amino acid side chainwhich serves to position a positively charged group in this position; Bis a non-aromatic amino acid, and C is any common or uncommon,hydrophobic amino acid side chain which serves to position any alkyl,aromatic or other group in this position; and D is any common oruncommon, aromatic amino acid which serve to position an aromaticside-chain in this position, and has the structure:

where Z is indole, indole methyl, benzyl, benzene, naphthyl, naphthylmethyl, or a derivative thereof; and R is H or an alkyl, aromatic, acylor aromatic-acyl group; E is any amino acid other than tryptophan andhomotryptophan, and F is the side chain of a D- or L-amino acid selectedfrom the group consisting of arginine, homoarginine, lysine andhomolysine.
 35. A composition comprising a compound according to claim34, together with a pharmaceutically acceptable carrier or excipient.36. A method of agonizing the activity of a C5a receptor on a cellcomprising contacting the cell with the compound of claim 34 in anamount sufficient to agonize the C5a receptor on the cell.
 37. Themethod of claim 36, wherein the cell is in a mammal and said contactingcomprises administering the compound to said mammal.
 38. The method ofclaim 31, wherein the mammal is a human.
 39. A method of treatinginflammatory arthritis mediated by a C5a receptor, comprising the stepof administering an effective amount of a compound according to claim 34to a mammal in need thereof.
 40. The method of claim 39, wherein themammal is a human.
 41. The method of claim 39, wherein the inflammatoryarthritis is rheumatoid arthritis.
 42. A method of treating inflammatoryarthritis, comprising the step of administering an effective amount of acompound according to claim 34 to a mammal in need thereof.
 43. Themethod of claim 42, wherein the mammal is a human.
 44. The method ofclaim 42, wherein the inflammatory arthritis is rheumatoid arthritis.45. A compound having the formula


46. A composition comprising the compound of claim 45, together with apharmaceutically acceptable carrier or excipient.
 47. A method ofagonizing the activity of a C5a receptor on a cell comprising contactingthe cell with the compound of claim 45 in an amount sufficient toagonize the C5a receptor on the cell.
 48. The method of claim 47,wherein the cell is in a mammal and said contacting comprisesadministering the compound to said mammal.
 49. The method of claim 48,wherein the mammal is a human.
 50. A method of treating inflammatoryarthritis, comprising the step of administering an effective amount of acompound according to claim 45 to a mammal in need thereof.
 51. Themethod of claim 50, wherein the mammal is a human.
 52. A method oftreating inflammatory arthritis mediated by a C5a receptor, comprisingthe step of administering an effective amount of a compound according toclaim 45 to a mammal in need thereof.
 53. The method of claim 52,wherein the mammal is a human.
 54. The method of claim 52, wherein theinflammatory arthritis is rheumatoid arthritis.